There are a number of reasons to consider using streptavidin monolayer crystals as “affinity” support films for cryo-electron microscopy (cryo-EM). Macromolecules of interest can be easily tagged with biotin or a streptavidin-binding peptide and then bound to streptavidin (SA) with high affinity and specificity. Furthermore, tagging followed by affinity binding is expected to pose less risk to the native structure of the macromolecule than does (1) adsorption of particles to the surface of carbon film, even when rendered hydrophilic by exposure to a glow discharge, or (2) repeated collision with the air-water interface that occurs when freely diffusing macromolecules are confined to a thin aqueous film (Taylor and Glaeser, 2008).
Monolayer crystals of SA have been considered previously by several authors for use as an affinity-support film. One early study viewed SA as being a “general adaptor” for linking any kind of biotinylated molecule to a lipid monolayer (Darst et al., 1991). Chemically biotinylated ferritin was used in that work to show that a high density of randomly distributed particles could be bound to 2-D crystals of SA. In an extension of the adaptor-molecule idea, Crucifix et al. first randomly decorated SA monolayer crystals with biotinylated dsDNA molecules, and then used the immobilized DNA as bait to bind yeast RNA Pol I particles (Crucifix et al., 2004). Wang et al. showed that biotinylated proteoliposomes could be bound at high density (Wang and Sigworth, 2009; Wang et al., 2008), and they introduced the further innovation of eliminating the periodic background due to SA by masking out the Bragg reflections in the computed Fourier transforms of images. Han et al. then went on to demonstrate the generality with which chemical biotinylation of soluble-protein complexes could be used (Han et al., 2012).
In spite of these promising demonstrations, SA monolayer crystals have not been adopted as support films for routine data collection. Two major problems remained with the use of SA monolayer crystals. First, the standard protocol for growing monolayer crystals involves an additional incubation step of 2 hours or more (Wang and Sigworth, 2010), which both slows and complicates the process of preparing cryo-EM specimens. Second, while the results can be excellent, the growth of large, well-ordered crystals on micro-wells, together with their transfer onto EM grids, is quite inconsistent.